U.S. patent application number 12/678636 was filed with the patent office on 2010-11-11 for solid phase cell isolation and/or enrichment method.
This patent application is currently assigned to AdnaGen AG. Invention is credited to Winfried Albert, Siegfried Hauch.
Application Number | 20100285581 12/678636 |
Document ID | / |
Family ID | 38659627 |
Filed Date | 2010-11-11 |
United States Patent
Application |
20100285581 |
Kind Code |
A1 |
Hauch; Siegfried ; et
al. |
November 11, 2010 |
Solid Phase Cell Isolation and/or Enrichment Method
Abstract
The present invention concerns a solid phase method for
isolating and/or enriching predetermined cells from a sample. Such
methods are used e.g. to isolate and enrich predetermined cells
like fetal cells from a sample of maternal peripheral blood, tumor
cells from a sample of body fluid or stem cells from a fluid or
fluidized sample of body tissue or body fluid. The solid phase
isolation method of the present invention is used for isolating
predetermined cells from a sample containing such predetermined
cells by binding the predetermined cells to a solid surface.
According to the invention the sample is contacted with the solid
surface and then removed from the solid surface, wherein the sample
or a washing buffer contains a polyol during or after contacting
the sample with the solid surface.
Inventors: |
Hauch; Siegfried;
(Coppenbrugge, DE) ; Albert; Winfried; (Penzberg,
DE) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI L.L.P.
600 CONGRESS AVE., SUITE 2400
AUSTIN
TX
78701
US
|
Assignee: |
AdnaGen AG
Hannover-Langenhagen
DE
|
Family ID: |
38659627 |
Appl. No.: |
12/678636 |
Filed: |
September 17, 2008 |
PCT Filed: |
September 17, 2008 |
PCT NO: |
PCT/EP2008/007774 |
371 Date: |
July 27, 2010 |
Current U.S.
Class: |
435/366 ;
435/378 |
Current CPC
Class: |
G01N 33/54393 20130101;
C12N 2500/35 20130101; G01N 33/574 20130101; G01N 1/34
20130101 |
Class at
Publication: |
435/366 ;
435/378 |
International
Class: |
C12N 5/071 20100101
C12N005/071; C12N 5/078 20100101 C12N005/078; C12N 5/09 20100101
C12N005/09; C12N 5/073 20100101 C12N005/073 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 17, 2007 |
EP |
07018205.0 |
May 9, 2008 |
EP |
08008770.3 |
Claims
1. A solid phase isolation method for isolating predetermined cells
from a sample containing the predetermined cells comprising
contacting the predetermined cells with a solid surface, and then
washing the solid surface, wherein the sample contains a polyol at
least during one of (a) contacting the sample with the solid
surface or (b) washing.
2. The method according to claim 1, wherein before or during
contacting the sample with the solid surface, the polyol is added
to the sample.
3. The method according to claim 1, wherein a washing buffer for
washing the solid surface contains the polyol.
4. The method according to claim 1, wherein the polyol is added to
the sample or contained in the washing buffer in a final
concentration (W/V or V/V) of at least 1%, preferably at least 10%,
preferably at least 20%, preferably at least 30%, preferably at
least 50%, preferably at least 60% polyol.
5. The method according to claim 1, wherein at least one of the
polyols selected from sorbitol, sucrose, trehalose, mannitol,
fructose, maltit, lactitol, xylitol and glycerol is added to the
sample.
6. The method according to claim 1, wherein the solid surface is
selected from a gel surface, sepharose surface, glass surface,
latex surface, ceramics surface, metal surface and plastic
surface.
7. The method according to claim 1, wherein the solid surface is
the surface of magnetic beads.
8. The method according to claim 1, wherein ligands or antibodies
are immobilized on the solid surface, which ligands or antibodies
specifically bind to the predetermined cells.
9. (canceled)
10. The method according to claim 1, wherein the predetermined
cells are rare cells.
11. The method according to claim 1, wherein the predetermined
cells are fetal cells, and the sample is maternal peripheral
blood.
12. The method according to claim 1, wherein the predetermined
cells are tumor cells from cell suspensions including at least one
of peripheral blood, bone marrow, urine, ascites, or sputum from a
patient.
13. The method according to claim 1, wherein the predetermined
cells are stem cells, and the sample is a body fluid and/or from a
tissue sample from a human being.
14. The method according to claim 1, wherein the predetermined
cells are tumor stem cells and/or EMT cells, and the sample is a
body fluid and/or from a tissue sample.
15. A solid phase enrichment method for enriching predetermined
cells from a sample containing the predetermined cells comprising
contacting the predetermined cells with a solid surface, and then
washing the solid surface wherein the sample contains a polyol at
least during one of (a) contacting the sample with the solid
surface or (b) washing.
16. The method according to claim 15, wherein before or during
contacting the sample with the solid surface, the polyol is added
to the sample.
17. The method according to claim 15, wherein a washing buffer for
washing the solid surface contains the polyol.
18. The method according to claim 15, wherein the predetermined
cells are fetal cells, and the sample is maternal peripheral
blood.
19. The method according to claim 15, wherein the predetermined
cells are tumor cells and the sample is a cell suspension including
at least one of peripheral blood, bone marrow, urine, ascites, or
sputum from a patient.
20. The method according to claim 1, wherein the predetermined
cells are stem cells, and the sample is a body fluid and/or from a
tissue sample from a human being.
21. The method according to claim 1, wherein the predetermined
cells are tumor stem cells and/or EMT cells, and the sample is a
body fluid and/or from a tissue sample.
Description
[0001] The present invention concerns a solid phase method for
isolating and/or enriching predetermined cells from a sample. Such
methods are used e.g. to isolate and enrich predetermined cells
like fetal cells from a sample of maternal peripheral blood, tumor
cells from a sample of body fluid or stem cells from a fluid or
fluidized sample of body tissue or body fluid.
[0002] The detection and analysis of rare cells in blood, bone
marrow and other specimens becomes an important need in
diagnostics: The detection of circulating tumor cells, including
tumor stem cells and stem cells in epithelial-mesenchymal
transition (EMT), and minimal residual cancer is particularly
useful in oncology for improved prognosis, early detection of
disease progression and therapy monitoring. Another application of
such techniques is the detection and analysis of fetal cells in
maternal blood that enables effective and non-invasive prenatal
diagnosis of genetic and chromosomal aberrations at early stages of
fetal development.
[0003] PCR based DNA analysis or RT-PCR based expression profiling
are sensitive and easy to handle technologies for the detection and
analysis of such rare cells. However, these technologies are
affected by the fact, that contaminating leukocytes as a source of
non-specific background signals due to inherent illegitimate
expression or endogenous expression of tumor associated antigens
are lowering the specificity of the detection of rare cells, e.g.
circulating tumor cells. Similarly, DNA genotyping is not possible
if the isolated tumor or fetal cells are not sufficiently pure.
[0004] The enrichment of cells from bodily specimens is an
important task in many therapeutic applications, too, besides
diagnostic applications, since monoclonal antibodies (or other
ligands and specifiers) are available now that allow the separation
of many particular cell types from sources with mixed cell
populations such as blood, bone marrow and other tissues. Cell
based therapies are a growing field that places serious demands on
the selectivity of any cell isolation technique that is useful for
that purpose. Stem cell graft engineering and immunotherapy of
cancer are one of the most important applications in cellular
therapy. Autologous stem cells enriched from blood or bone marrow
can be used to support high dose chemotherapy regimens for a
variety of malignancies.
[0005] There is a great demand for a high purity of cells isolated
with cell enrichment technologies:
[0006] The usefulness of enriched stem cell products is impaired by
contaminating malignant cells that might be a source for later
metastases and relapses. A contamination with T-cells is the major
cause of a graft versus host disease and is the primary reason for
transplantation failure. Thus, an improvement of the efficacy of
existing therapeutic cell enrichment technologies is a major issue
that has to be urgently addressed.
[0007] There are several techniques available for the enrichment of
cells from blood and other specimens. Cell sorting by FACS
technology is a very specific method that can be applied to
enumerate and collect rare cells for further use and analysis.
However, this method is not applicable yet routinely to whole blood
samples or large volumes and for large scale cell preparations.
Several immunochemical methods have been developed for the
enrichment of cells from fluid specimens using solid phase
adsorption as for instance by immunocapturing. Monoclonal
antibodies (or other adequate ligands like, for instance, aptamers
or other specifiers) can be immobilised on solid surfaces like
sepharose, glass, latex or plastic beads or other surfaces for
through-flow column or batch applications. Although the handling of
these devices is relatively convenient, recovery and purity
obtained with such devices is insufficient in many cases. The use
of antibody-labelled magnetic beads turned out to be quite
efficient, showing good recovery rates and an enrichment rate of
4-5 log.sub.10. Nevertheless, this purity is still too low for
expression profiling, especially if the molecular marker of
interest is not over-expressed in the target cells as compared to
potentially contaminating nucleated cells like leukocytes or
erythropoietic stem cells. Furthermore, DNA-based genetic typing
experiments to differentiate fetal from maternal cells or tumor
from normal cells are generally not possible in such samples.
Therefore, increasing efficacy of cell purification with solid
phase immunochemical or similar devices is an urgent need not only
for diagnostic but also for therapeutic applications.
[0008] There are several reasons for non-specific binding or
trapping of unwanted cells during cell enrichment:
[0009] 1. Although monoclonal antibodies (or other selected ligands
and specifiers) should exhibit a high specificity towards the
antigen (or receptor) chosen for cell separation, there may still
be considerable non-specific binding to similar antigen or receptor
structures.
[0010] 2. Another reason for trapping unwanted cells may be
physical interactions (hydrophobic or electrostatic interactions or
simply mechanical trapping) with and within the solid
phase/antibody (ligand or specifier) structure.
[0011] 3. The target antigens or receptors chosen for cell
enrichment might also be expressed on some non-target cells present
in the same specimen (e.g. through illegitimate or endogenous
expression).
[0012] In order to improve the performance of present days
enrichment methods, these methods could be adapted to obtain better
specificity by choosing a more specific antibody, ligand or
specifier towards a chosen cell surface target (see 1 and 2 above)
whereas non-specific carryover of non-target cells cannot be easily
prevented (see 3 above). Choosing magnetic beads as carriers for
antibodies, ligands or receptors instead of through-flow columns
filled with beads or choosing polystyrol beads instead of sepharose
are examples for possible improvements. However, most attempts for
technical improvements did not lead to the purities required in
many cases, and there is still an urgent need for a further
reduction of the amount of non-specifically bound or trapped
non-target cells, preferably by adjusting binding conditions that
avoid physical interactions.
[0013] It is the object of the present invention to provide an
improved isolation and/or enrichment method based on solid phase
technology, that successfully removes or diminishes
non-specifically bound or trapped non-target cells during
solid-phase capturing like immunocapturing or ligand-capturing.
[0014] This object is solved by the solid phase isolation and/or
enrichment method according to claim 1. Improvements of the
inventive method are given in claims 2 to 8. Further claims 9 to 14
provide usages of the solid phase isolation and/or enrichment
method according to the invention.
[0015] The present invention enables for the first time the use of
solid phase immuno- or ligand-capturing procedures for important
applications, e.g. genotype expression profiling, that require
purified cell populations obtained from mixed cell populations like
blood, bone marrow or similar specimens. The inventive method
successfully removes or diminishes non-specifically bound or
trapped non-target cells during solid phase isolation like solid
phase immunocapturing or solid phase ligand-capturing.
[0016] The use of polyols for protein stabilisation is generally
known and described. For example, glycerol is frequently used for
the cryo-conservation of cells, and other polyols like mannitol are
effectively used for the preservation of red blood cell
preparations. Polyols (e.g. sorbitol, sucrose, trehalose) are known
to be useful for protection of bacterial and eukaryotic cells
during drying or heat shock procedures.
[0017] Different to these known uses of polyols in the prior art,
the inventors observed to their great surprise, that polyols may
not only be used for preservation of samples but are also able to
reduce the background signals caused by non-specific bound or
trapped non-target cells dramatically when used in solid phase cell
enrichment procedures, whereas at the same time the recovery of
target cells was not affected. It can be speculated that
interactions of polyols with hydrophobic protein structures
increase the specifity of solid phase antigen/antibody (ligand or
specifier/receptor) interactions possibly by lowering non-specific
hydrophobic binding and by stabilizing the secondary and the
tertiary structures of antibodies, ligands or specifiers and
antigens or receptors, thereby enhancing the binding affinity and
reducing non-specific interactions.
[0018] By adding polyols to the sample during solid phase cell
enrichment procedures either during contacting the sample with the
solid phase or in a subsequent washing step of the solid phase, it
could be observed that the amount of non-specifically bound or
trapped non-target cells was reduced by 99%, e.g. when using an
inventive 50% (V/V) glycerol/PBS washing buffer in a manner as
described e.g. in the Manual of the AdnaTest BreastCancerSelect
(AdnaGen AG, Langenhagen, Germany) using antibodies against
epithelial and tumor-associated antigens (EPCAM, MUC1, HER2)
conjugated to magnetic beads. It further turned out that the
recovery of target cells was not affected and the concentration of
the target cells was very low. The addition of polyols, preferably
glycerol, to samples before the cell enrichment procedure decreased
the non-specifically bound or trapped non-target cell load
substantially. This could also be observed at lower concentrations
of glycerol, e.g. 1 to 10% (V/V). In the following, some examples
of the inventive method are given.
[0019] In these examples,
[0020] FIG. 1 shows a schematic overview of the sample preparation
and analysis;
[0021] FIG. 2 shows the effect of different glycerol concentrations
in the AdnaTest BreastCancerSelect washing buffer on CD45 mRNA
levels;
[0022] FIG. 3 shows in FIG. 3a the effect of a washing buffer
containing 20% glycerol and 5% mannitol on the recovery of 2 MCF7
tumor cells spiked into 5 ml blood of healthy donors and FIG. 3b
the decrease of CD45 mRNA levels due to the modified washing
buffer;
[0023] FIG. 4 shows the effect of glycerol added to blood samples
on the non-specific binding of leukocytes;
[0024] FIG. 5 shows 5 healthy donor blood samples, analyzed in
duplicates for estrogen receptor (ER) expression with the AdnaTest
BreastCancerDetect followed by PCR amplification of ER cDNA
developed for this purpose. ER was amplified in all samples if PBS
only was used during the bead washing step. The average amplicon
concentrations were 0.15 ng/.mu.l. This non-specific background
activity disappeared when PBS/glycerol was used for washing;
[0025] FIG. 6 shows the influence of different polyols in the
washing buffer on leukocyte background;
[0026] FIG. 7 shows the dependency of the purity of CD34(+)-stem
cells purified through anti-CD34 antibody coated magnetic beads on
the glycerol concentration in the washing buffer; and
[0027] FIG. 8 shows the effect of polyols on leucocyte background
for the isolation of tumor stem cells and EMT cells and subsequent
detection of the relevant marker expression.
[0028] Examples 1-4 have been performed according to the
manufacturers instructions for the detection of CTC.
EXAMPLE 1
[0029] 5 ml blood samples obtained from healthy donors were
processed with the AdnaTest BreastCancerSelect followed by a
subsequent determination of CD45 mRNA expression. After the
inoculation of the blood samples with the magnetic beads of the
AdnaTest BreastCancerSelect, the subsequent washing steps were
performed with PBS buffer without or with addition of different
amounts of glycerol (0-50% (V/V)) (see FIG. 2). The CD45 mRNA
levels decrease with increasing glycerol concentrations as shown in
FIG. 1. This indicates the disappearance of contaminating
leukocytes as the source of the CD45 mRNA expression.
EXAMPLE 2
[0030] 2 MCF7 breast cancer cells were spiked into 5 ml blood drawn
from healthy donors. The tumor cells recovered with the AdnaTest
BreastCancerSelect were subsequently analysed for tumor associated
mRNA markers using the AdnaTest BreastCancerDetect. This was
followed by a CD45 PCR to estimate the decrease of the leukocyte
contamination. After the inoculation of the blood samples with the
AdnaTest BreastCancerSelect magnetic beads, the washing steps were
performed with PBS containing 20% (V/V) of glycerol and 5 (W/V)
mannitol. As shown in FIG. 3a, the recovery of the spiked cells was
not impaired by the modified washing buffer. Surprisingly, the CD45
and actin mRNA concentrations decreased at the same time as the
amplicon concentrations of the tumor associated markers increased,
indicating a reduction of contaminating leukocytes and a higher
yield of tumor cell mRNA (FIG. 3b).
EXAMPLE 3
[0031] Glycerol was added in different concentrations (0-1% (V/V))
to 5 ml blood samples from healthy donors. The samples were
analysed using the AdnaTest BreastCancerSelect/Detect followed by a
subsequent determination of CD45 mRNA expression. The CD45 amount
is decreasing with increasing glycerol concentrations indicating a
lower amount of bound or trapped leukocytes as shown in FIG. 4.
[0032] RT-PCR assay addressing the expression of estrogen receptor
(ER) and progesterone receptor (PR) in circulating tumor cells was
developed for inclusion in the AdnaTest BreastCancerSelect/Detect.
Since the test for ER expression shows a relatively high background
due to bound or trapped leukocytes expressing ER, it is unable to
surpass a specificity of 80% at the required analytical sensitivity
level (i.e. 1 or 2 tumor cells in 5 ml blood). This background
activity (about 0.15 ng/.mu.l on average), responsible for the
reduction of the specificity, can be eliminated if PBS containing
30% (V/V) glycerol is used in the washing steps as shown in FIG.
5.
EXAMPLE 4
[0033] The effect of different polyols in the washing buffer on
CD45 expression in the blood of healthy donors was determined in
order to show the ability of different polyols to minimize
unspecific background.
[0034] 5 ml blood samples obtained from healthy donors were
processed with AdnaTestBreastCancer Select followed by a CD45
RT-PCR. The washing steps were performed with PBS buffer containing
one of said three polyols (sorbitol (10%, W/V), fructose (10% W/V),
glycerol (10% V/V)). PBS buffer without additive was used as a
control and for an additional final wash in all samples before cell
lysis and RT-PCR. Detection of CD45 expression is an indicator for
selectivity in the separation step followed by detection of CD45 as
marker for residual leukocyte cells. As shown in FIG. 6, all
polyols caused a reduction of CD45. Sorbitol and fructose caused
about 15% and glycerol about 35% reduction of leukocyte background.
Obviously, as shown with these three arbitrarily selected polyols,
all polyols are suitable for the present invention.
EXAMPLE 5
[0035] In this example, the purity of CD34-positive stem cells from
cord blood after immunomagnetic enrichment is determined depending
on the glycerol concentration in the washing buffer (see FIG.
7).
[0036] Mononuclear cells (MNC), 3.48.times.10.sup.8 cells;
9.7.times.10.sup.8 cells and 2.59.times.10.sup.8 cells, obtained
from cord blood were re-suspended in 1 ml PBS buffer containing
various concentrations of glycerol (V/V), 5% (W/V) mannitol and
0.1% BSA. After adding magnetic beads (Dynal) with anti-CD34
antibodies coupled to them, the suspension was incubated for 30 min
in an overhead shaker at room temperature.
[0037] After incubation, the beads cell suspension was washed 3
times with 1 ml PBS buffer containing various concentrations of
glycerol (V/V) and 5% (W/V) mannitol and 0.1% (W/V) BSA followed by
lysis of the bead cell complexes in 200 .mu.l lysis buffer (Dynal).
After mRNA isolation and reverse transcription, the resulting cDNA
was analyzed by PCR for CD45 (to determine trapped leukocytes) and
CD34 (to determine enriched stem cells) transcripts. A ratio of the
quantified PCR signal was calculated to determine the relative
purity of the stem cells in relation to the glycerol concentration
in the washing buffer.
[0038] As is shown in FIG. 7, glycerol/mannitol containing PBS
washing buffers significantly increase the purity of the CD34
fraction (stem cell fraction) with increasing glycerol
concentration in the washing buffer compared to the washing buffer
without any polyol.
EXAMPLE 6
[0039] The detection of EMT and tumor cell markers is impeded by
the high background signals produced by contaminating
leukocytes.
[0040] To determine the effect of the AdnaWash buffer, containing
the polyols glycerol (23% (V/V)) and mannitol (5% (W/V)) in PBS,
healthy donor samples were processed with the AdnaTest
BreastCancerSelect reagents according to instruction with and
without addition of this buffer. The cDNA obtained from these
samples was analyzed by PCR for the EMT markers PI3KCA, SIP1 and
Akt2 as well as for the tumor stem cell markers ALDH1 and BMI1.
[0041] As shown in FIG. 8, polyols decrease the leukocyte signals
interfering with EMT markers (Akt2, PI3KCA, SIP1) and tumor stem
cell markers (BMI1, ALDH1) analysis due to removal of trapped
leukocytes which is confirmed by the decrease of the actin
signal.
[0042] By this example it is shown that trapped leukocytes express
EMT and stem cell markers and produce unacceptable strong
background signals. These signals could be efficiently reduced with
a polyol containing washing buffer enabling a specific analysis of
these markers on CTC. However, recovery of the CTC was not
reduced.
* * * * *